Erratum in

Abstract

INTRODUCTION:

Bcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy.

METHODS:

Four human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice.

RESULTS:

Treatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells.

CONCLUSION:

Targeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer.

Expression levels of Bcl-2 and Bcl-xL proteins in MDA-MB-231, MDA-MD-453, BT-474, and ZR-75-1 cells. (a) Western blot analysis of Bcl-2 and Bcl-xL expression. (b) Quantification of Bcl-2 and Bcl-xL expression by densitometric analysis. The relative expression of Bcl-2 and Bcl-xL in MDA-MB-453 cells was compared with the expression in MDA-MB-231, BT-474, and ZR-75-1 cells. Results are from two representative, independent experiments.

Sequence-specific downregulation and cytotoxic effects of antisense Bcl-2 oligodeoxynucleotides on BT-474 and ZR-75-1 cells. (a) Specific inhibition of Bcl-2 protein expression by treatment with antisense (AS) Bcl-2 oligodeoxynucleotides (ODNs). Cells were treated with 10 μg/ml Lipofectamine alone (control) or 1.0 μM AS, mismatch control (MM), or random control (RC) ODNs for 24 hours. Cells were then cultured in standard medium, total protein was extracted, and Bcl-2 and β-actin protein levels were analyzed by Western blotting. (b) Quantification of Bcl-2 expression by densitometric analysis. The expression of Bcl-2 in cells treated with control, AS Bcl-2, RC Bcl-2, and MM Bcl-2 ODNs was normalized with β-actin, and the relative values are presented. Error bars indicate SD. The data presented are from three independent experiments. (c) Effects of AS Bcl-2 ODNs on the proliferation of BT-474 and ZR-75-1 breast cancer cells in vitro. Cells were treated with various concentrations of AS Bcl-2 ODNs in 24-well dishes. Four days after treatment, cells were stained with trypan blue and counted. Error bars indicate SD. The data presented are from three independent experiments.

Sequence-specific downregulation and cytotoxic effects of antisense Bcl-xL oligodeoxynucleotides on MDA-MB-231 and BT-474 cells. (a) Specific inhibition of Bcl-xL protein expression by treatment with antisense (AS) Bcl-xL oligodeoxynucleotides (ODNs). Cells were treated with 10 μg/ml Lipofectamine alone (control) or 1.0 μM AS, mismatch control (MM), or random control (RC) ODNs for 24 hours. Cells were then cultured in standard medium, total protein was extracted, and Bcl-xL and β-actin protein levels were analyzed by Western blotting. (b) Quantification of Bcl-xL protein expression by densitometric analysis. The Bcl-xL protein expression was normalized with β-actin, and the relative values are presented. Error bars indicate SD. The data presented are from three independent experiments. (c) Effects of AS Bcl-xL ODNs on the proliferation of MDA-MB-231 and BT-474 breast cancer cells in vitro. Cells were treated with various concentrations of AS Bcl-xL ODNs in 24-well dishes. Four days after treatment, cells were stained with trypan blue and counted. Error bars indicate SD. The data presented are from three independent experiments.

Effect of treatment of ZR-75-1 cells with antisense Bcl-2 and doxorubicin on the apoptosis-related proteins. Cells were pretreated with 10 μg/ml Lipofectamine alone, or 1.0 μM antisense Bcl-2 oligodeoxynucleotides, for 24 hours. Cells were then cultured in standard medium, after which they were treated with 0.5 μM doxorubicin (DOX) for 6, 12, or 24 hours. Whole cell lysate was then extracted and subjected to Western blotting. The data presented are from more than two independent experiments.

Effects of treatment with antisense Bcl-xL and mitomycin C, doxorubicin, paclitaxel, or docetaxel on MDA-MB-231 cells. (a) Expression levels of Bcl-xL and Bcl-2 protein in MDA-MB-231 cells transplanted into athymic mice after treatment with antisense (AS) Bcl-xL oligodeoxynucleotides (ODNs) were measured by Western blot analysis at the indicated time points. (b) Enhancement of the antitumor effects of anticancer drugs by AS Bcl-xL ODNs in MDA-MB-231 tumor xenografts. Each point represents the mean tumor volume of the four mice in each group. Error bars indicate SD. *, P < 0.05, analysis of variance with Fisher's least significant difference test. The data presented are from two independent experiments. MMC, mitomycin C; DOX, doxorubicin; TXL, paclitaxel; TXT, docetaxel.

Effect of antisense Bcl-2 oligodeoxynucleotides on splenomegaly development and serum IL-12 levels in athymic mice. (a) Segment of spleen from a mouse with splenomegaly after treatment with antisense (AS) Bcl-2 oligodeoxynucleotides (ODNs), and segments from untreated (control) and synthetic CpG AS Bcl-2 ODN-treated mice. (b) Comparison of splenic weight among mice treated with AS Bcl-2 ODNs, synthetic CpG AS Bcl-2 ODNs, and control mice. (c) Increased serum levels of IL-12 were observed in mice treated with AS Bcl-2 ODNs, compared with those treated with synthetic CpG AS Bcl-2 ODNs, and with control mice. Each point represents the mean of the three mice in each group. Error bars indicate SD. The data presented are from three independent experiments.

Effect of synthetic CpG antisense Bcl-2 on BT-474 cells in comparison with antisense Bcl-2. Each point represents the mean tumor volume of the four mice in each group. Error bars indicate SD. The data presented are from two independent experiments. RC, random control; TXT, docetaxel.